Staphylococcus aureus is a highly virulent and widely successful pathogen, which is speculated to be the most common cause of human disease. Currently, it is the leading agent of nosocomial infections worldwide, causing a variety of ailments in a plethora of ecological niches within the host. S. aureus is also a major public health concern due to the continued emergence of multi-drug resistant isolates (MRSA), including those resistant to last resort glycopeptide antibiotics (VRSA). In addition to this, over the last 10 years, there has been a disturbing and meteoric increase in cases of severe invasive S. aureus infections in healthy subjects, lacking any predisposing factors or connections to healthcare settings. Apparently, hypervirulent strains of MRSA have evolved in the community (CA-MRSA), possessing unique combinations of virulence factors and resistance traits. The reason for the enhanced virulence and transmission of CA-MRSA strains is unclear;however recent work suggests that it may be the result of hypersecretion of agr regulated toxins. Included amongst these are extracellular proteases, which ourselves and others have shown to be overproduced in CA-MRSA strains compared to HA-MRSA isolates. Despite reports by our group demonstrating the contribution of extracellular proteases to disease causation, their role as key virulence determinants in S. aureus infections is still unclear. Therefore, given the current clinical significance of CA-MRSA infections, and the fact that these strains hyper secrete extracellular proteases, we propose to definitively explore the contribution of these enzymes to S. aureus pathogenesis. To achieve this we will: 1. Investigate the role of extracellular proteases in S. aureus physiology and virulence determinant stability. This will be carried out by: i) Generating a derivative of the leading CA-MRSA strain (USA300) in which each of the 4 major protease loci has been deleted. This strain will then be use to: ii) Phenotypically characterize the in vitro contribution of these enzymes to S. aureus growth and physiology;and iii) Explore the effect of exoproteases on virulence determinant stability using proteomics to analyze alterations in secreted and cell wall proteomes. Having determined the impact of the extracellular proteases in vitro we will then use our mutant strain to assess their contribution to virulence in vivo. To achieve this we will: 2. Explore the impact of extracellular proteases on CA-MRSA pathogenesis. This will be carried out by: i) Assessing the contribution of extracellular proteases to S. aureus skin and soft tissue infections;and ii) Exploring the role of these enzymes in severe invasive disease using a murine model of sepsis and dissemination. PUBLIC HEALTH RELEVANCE: Staphylococcus aureus is a highly virulent and widely successful pathogen that is speculated to be the most common cause of human infection. With the continued emergence of multi-drug resistant isolates of S. aureus (such as MRSA), there is an urgent need to understand the mechanisms by which this deadly pathogen causes disease. This proposal seeks to understand the contribution of secreted proteases to S. aureus disease causation.